The invention relates to novel targeting magnetic resonance imaging contrast agents and methods of detecting physiological signals or substances.
Magnetic resonance imaging (MRI) is a diagnostic and research procedure that uses high magnetic fields and radio-frequency signals to produce images. The most abundant molecular species in biological tissues is water. It is the quantum mechanical xe2x80x9cspinxe2x80x9d of the water proton nuclei that ultimately gives rise to the signal in all imaging experiments. In MRI the sample to be imaged is placed in a strong static magnetic field (1-12 Tesla) and the spins are excited with a pulse of radio frequency (RF) radiation to produce a net magnetization in the sample. Various magnetic field gradients and other RF pulses then act on the spins to code spatial information into the recorded signals. MRI is able to generate structural information in three dimensions in relatively short time spans.
The Image
MR images are typically displayed on a gray scale with black the lowest and white the highest measured intensity (I). This measured intensity I=C*M, where C is the concentration of spins (in this case, water concentration) and M is a measure of the magnetization present at time of the measurement. Although variations in water concentration (C) can give rise to contrast in MR images, it is the strong dependence of the rate of change of M on local environment that is the source of image intensity variation in MRI. Two characteristic relaxation times, T1 and T2, govern the rate at which the magnetization can be accurately measured. T1 is the exponential time constant for the spins to decay back to equilibrium after being perturbed by the RF pulse. In order to increase the signal-to-noise ratio (SNR) a typical MR imaging scan (RF and gradient pulse sequence and data acquisition) is repeated at a constant rate for a predetermined number of times and the data averaged. The signal amplitude recorded for any given scan is proportional to the number of spins that have decayed back to equilibrium since the previous scan. Thus, regions with rapidly decaying spins (i.e. short T1 values) will recover all of their signal amplitude between successive scans.
The measured intensities in the final image will accurately reflect the spin density (i.e. water content). Regions with long T1 values compared to the time between scans will progressively lose signal until a steady state condition is reached and will appear as darker regions in the final image. Changes in T2 (spin-spin relaxation time) result in changes in the signal linewidth (shorter T2 values) yielding larger linewidths. In extreme situations the linewidth can be so large that the signal is indistinguishable from background noise. In clinical imaging, water relaxation characteristics vary from tissue to tissue, providing the contrast which allows the discrimination of tissue types. Moreover, the MRI experiment can be setup so that regions of the sample with short T1 values and/or long T2 values are preferentially enhanced so called T1-weighted and T2-weighted imaging protocol.
MRI Contrast Agents
There is a rapidly growing body of literature demonstrating the clinical effectiveness of paramagnetic contrast agents (currently 8 are in clinical trials or in use). The capacity to differentiate regions/tissues that may be magnetically similar but histologically distinct is a major impetus for the preparation of these agents. In the design of MRI agents, strict attention must be given to a variety of properties that will ultimately effect the physiological outcome apart from the ability to provide contrast enhancement. Two fundamental properties that must be considered are biocompatability and proton relaxation enhancement. Biocompatability is influenced by several factors including toxicity, stability (thermodynamic and kinetic), pharmacokinetics and biodistribution. Proton relaxation enhancement (or relaxivity) is chiefly governed by the choice of metal and rotational correlation times.
The first feature to be considered during the design stage is the selection of the metal atom, which will dominate the measured relaxivity of the complex. Paramagnetic metal ions, as a result of their unpaired electrons, act as potent relaxation enhancement agents. They decrease the T1 and T2 relaxation times of nearby (r6 dependence) spins. Some paramagnetic ions decrease the T1 without causing substantial linebroadening (e.g. gadolinium (III), (Gd3+)), while others induce drastic linebroadening (e.g. superparamagnetic iron oxide). The mechanism of T1 relaxation is generally a through space dipole-dipole interaction between the unpaired electrons of the paramagnet (the metal atom with an unpaired electron) and bulk water molecules (water molecules that are not xe2x80x9cboundxe2x80x9d to the metal atom) that are in fast exchange with water molecules in the metal""s inner coordination sphere (are bound to the metal atom).
For example, regions associated with a Gd3+ ion (near-by water molecules) appear bright in an MR image where the normal aqueous solution appears as dark background if the time between successive scans in the experiment is short (i.e. T1 weighted image). Localized T2 shortening caused by superparamagnetic particles is believed to be due to the local magnetic field inhomogeneities associated with the large magnetic moments of these particles. Regions associated with a superparamagnetic iron oxide particle appear dark in an MR image where the normal aqueous solution appears as high intensity background if the echo time (TE) in the spin-echo pulse sequence experiment is long (i.e. T2-weighted image). The lanthanide atom Gd3+ is by the far the most frequently chosen metal atom for MRI contrast agents because it has a very high magnetic moment (u2=63 BM2), and a symmetric electronic ground state, (S8). Transition metals such as high spin Mn(II) and Fe(III) are also candidates due to their high magnetic moments.
Once the appropriate metal has been selected, a suitable ligand or chelate must be found to render the complex nontoxic. The term chelator is derived from the Greek word chele which means a xe2x80x9ccrabs clawxe2x80x9d, an appropriate description for a material that uses its many xe2x80x9carmsxe2x80x9d to grab and hold on to a metal atom (see DTPA below). Several factors influence the stability of chelate complexes include enthalpy and entropy effects (e.g. number, charge and basicity of coordinating groups, ligand field and conformational effects). Various molecular design features of the ligand can be directly correlated with physiological results. For example, the presence of a single methyl group on a given ligand structure can have a pronounced effect on clearance rate. While the addition of a bromine group can force a given complex from a purely extracellular role to an effective agent that collects in hepatocytes.
Diethylenetriaminepentaacetic (DTPA) chelates and thus acts to detoxify lanthanide ions. The stability constant (K) for Gd(DTPA)2xe2x88x92 is very high (logK=22.4) and is more commonly known as the formation constant (the higher the logK, the more stable the complex). This thermodynamic parameter indicates the fraction of Gd3+ ions that are in the unbound state will be quite small and should not be confused with the rate (kinetic stability) at which the loss of metal occurs (kf/kd). The water soluble Gd(DTPA)2xe2x88x92 chelate is stable, nontoxic, and one of the most widely used contrast enhancement agents in experimental and clinical imaging research. It was approved for clinical use in adult patients in June of 1988. It is an extracellular agent that accumulates in tissue by perfusion dominated processes.
To date, a number of chelators have been used, including diethylenetriaminepentaacetic (DTPA), 1,4,7,10-tetraazacyclododecanexe2x80x2-N,Nxe2x80x2Nxe2x80x3,Nxe2x80x2xe2x80x3-tetracetic acid (DOTA), and derivatives thereof. See U.S. Pat. Nos. 5,155,215, 5,087,440, 5,219,553, 5,188,816, 4,885,363, 5,358,704, 5,262,532, and Meyer et al., Invest. Radiol. 25: S53 (1990).
Image enhancement improvements using Gd(DTPA) are well documented in a number of applications (Runge et al., Magn, Reson. Imag. 3:85 (1991); Russell et al., AJR 152:813 (1989); Meyer et al., Invest. Radiol. 25:S53 (1990)) including visualizing blood-brain barrier disruptions caused by space occupying lesions and detection of abnormal vascularity. It has recently been applied to the functional mapping of the human visual cortex by defining regional cerebral hemodynamics (Belliveau et al., (1991) 254:719).
Another chelator used in Gd contrast agents is the macrocyclic ligand 1,4,7,10-tetraazacyclododecane-N,Nxe2x80x2,Nxe2x80x3Nxe2x80x2xe2x80x3-tetracetic acid (DOTA). The Gd-DOTA complex has been thoroughly studied in laboratory tests involving animals and humans. The complex is conformationally rigid, has an extremely high formation constant (logK=28.5), and at physiological pH possess very slow dissociation kinetics. Recently, the GdDOTA complex was approved as an MRI contrast agent for use in adults and infants in France and has been administered to over 4500 patients.
Previous work describes a new class of MRI contrast agents that report on physiologic or metabolic processes within a biological or other type of sample. See U.S. Pat. Nos. 5,707,605 and 5,980,862.
However, it would be desirable to target these functional MRI agents to tissues or locations to allow for better imaging. Accordingly, it is an object of the present invention to provide targeted MRI contrast or enhancement agents which allow the visualization and detection of physiological agents within an animal, tissue or cells.
In accordance with the above objects, the present invention provides MRI agent compositions comprising a first Gd(III) ion bound to a first chelator such that the Gd(III) ion has coordination atoms in at least 7 coordination sites of said Gd(III) ion and a first blocking moiety covalently attached to the first chelator which hinders the rapid exchange of water in the remaining coordination sites of the first Gd(III) ion. The blocking moiety is capable of interacting with a target substance such that the exchange of water in the remaining coordination sites of said first Gd(III) ion is increased. The agents further comprise at least a first targeting moiety. The agents optionally can comprise a second Gd(III) ion bound to a second chelator such that the second Gd(III) ion has coordination atoms in at least 7 coordination sites of the Gd(III) ion and a second blocking moiety covalently attached to the second chelator which hinders the rapid exchange of water in the remaining coordination sites of the second Gd(III) ion.
In an additional aspect, the invention provides MRI agents comprise chelators linked together using polymers.
In a further aspect, the invention provides MRI agents having the formula: 
wherein
M is a paramagnetic metal ion selected from the group consisting of Gd(III), Fe(III), Mn(II), Yt(III), Cr(III) and Dy(III);
A, B, C and D are selected from the group consisting of single bonds or double bonds;
X1, X2, X3 and X4 are xe2x80x94OH, xe2x80x94COOxe2x80x94, xe2x80x94CH2OH xe2x80x94CH2COOxe2x80x94, a blocking moiety or a targeting moiety;
R1-R12 are each a substitution group;
wherein at least one of X1-X4 and R1-R12 is a blocking moiety and wherein at least one of X1-X4 and R1-R12 is a targeting moiety.
In an additional aspect, the present invention provides MRI agent compositions having the formula: 
wherein
M is a paramagnetic metal ion selected from the group consisting of Gd(III), Fe(III), Mn(II), Yt(III), Cr(III) and Dy(III);
X5, X6, X7, X8 and X9 are xe2x80x94OH, xe2x80x94COOxe2x80x94, xe2x80x94CH2OH xe2x80x94CH2COOxe2x80x94, a blocking moiety or a targeting moiety;
R14-R22 are each a substitution group;
wherein at least one of X5-X9 and R14-R22 is a blocking moiety and wherein at least one of X5-X9 and R14-R22 is a targeting moiety.
In a further aspect, the invention provides pharmaceutical compositions comprising an MRI agent of the invention and a pharmaceutically acceptable carrier.
In an additional aspect, the present invention provides methods of magnetic resonance imaging of a cell, tissue or patient comprising administering an MRI agent of the invention to a cell, tissue or patient and rendering a magnetic resonance image of the cell, tissue or patient.